PROJECT SUMMARY The timing of DNA replication is a critical parameter of cellular growth. It correlates with patterns of transcriptional regulation, chromatin modification, chromosome structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of chromosome metabolism. A major impediment to understand the regulation of replication timing in the human genome has been the lack of robust assays for identifying the location and firing times of human replication origins. Current approaches suffer from low signal-to-noise ratios and poor concordance between independent laboratories. Moreover, ensemble techniques are unable to probe the coordination of origin firing, a subject significant interest in the field, because it has been proposed as a key factor in replication timing and efficiency. We propose to apply two new high-throughput single-molecule approaches that we have developed?Optical Replication Mapping and SMRT Repli-seq?to map replication origins and replication fork progression across the human genome. We will use replication profiles that we obtain to develop hypotheses about fundamental aspects of genome biology, such as such as how replication and transcription are coordinated, if the location of replication termination sites are regulated and how forks navigate difficult-to-replicate sequences. Successful completion of this work will elucidate the regulation of replication timing across the human genome, allow for the characterization of the sequence and epigenetic determinants for origin function, and provide robust origin maps and replication profiles for others to use. Moreover, dissemination of this technology will change the questions that biologists are able to ask about the regulation of DNA replication timing and its repercussions in diverse fields, such as development, chromatin biology, and epigenetics.